The use of transgenic plants for the production of pharmaceutical proteins and industrial enzymes has been proposed. In general, expression of recombinant proteins relies on stable integration of a heterologous gene into a host plant genome using, e.g., Agrobacterium-mediated. transformation or particle bombardment. In terms of cost, production of commercially valuable proteins using crops in the field is more competitive than other biological production systems (such as yeast, bacteria, or mammalian cell cultures) which require complex and high-maintenance bioreactors. Further, protein production in plant can be easily scaled-up to produce large quantities.
The invention is based on the discovery of a new promoter derived from a rice gene encoding for a glycine-rich RNA binding protein (designated OsGRP-A1). The OsGRP-A1 promoter can be used to express proteins in a plant and its tissues and organs, especially in the seedling, embryo, or sprout tissues. The sequence of OsGRP-A1 gene is shown below:
As indicated above, the complete nucleotide sequence of OsGRP-A1 is designated SEQ ID NO:3, while the complete amino acid sequence of the protein encoded by OsGRP-A1 is designated SEQ ID NO:4. The upper case nucleotides represent the cDNA sequence, while the lower case nucleotides represent the genomic sequences flanking the cDNA and in the intron. Nucleotide numberings are relative to the transcription start site. The putative TATA box is underlined. The OsGRP-A1 protein contains two consensus RNA binding domains (amino acids 10-15 and 49-56 of SEQ ID NO:4), which are shown in italics. A minimal promoter sequence from xe2x88x92180 to xe2x88x921 is designated SEQ ID NO:1 (bolded sequence), a larger promoter sequence from xe2x88x92500 to xe2x88x921 is designated SEQ ID NO:2, and the promoter-containing genomic sequence upstream of the transcriptional start site (from xe2x88x921730 to xe2x88x921) is designated SEQ ID NO:5.
This new promoter was shown to direct expression of a heterologous protein in transgenic rice seeds and embryos during germination and in cultured rice suspension cells. Consequently, the promoter can be used to regulate and direct expression of a heterologous protein or RNA (e.g., an antisense RNA) in transgenic plants, organs thereof, or in plant cells.
Accordingly, the invention features an isolated nucleic acid including SEQ ID NO:1 (e.g., SEQ ID NO:2), a promoter that hybridizes under stringent conditions to SEQ ID NO:1, or a promoter that is at least 50% (e.g., at least 60, 70, 80, 90, or 95%) identical to SEQ ID NO:1. The nucleic acid of the invention can further include a heterologous sequence to which a promoter containing SEQ ID NO:1 is operably linked, i.e., the promoter directs transcription of the heterologous sequence. The invention also includes vectors and transformed cells harboring a nucleic acid of the invention, as well as transgenic plants (e.g., a transgenic sprout or seedling) whose genomic DNA contains a nucleic acid of the invention. The transgenic plant can be a monocot (e.g., rice) or dicot. The invention further features a method of producing a transgenic plant or organ of a plant (e.g., a seed) by stably introducing a nucleic acid of the invention into a plant cell, and culturing the plant cell under conditions sufficient for the plant cell to form a plant or organ of a plant.
The invention also features nucleic acids containing SEQ ID NO:5 or a fragment thereof. These fragments are at least 6 nucleotides in length, e.g., at least 10, 15, 20, 50, 100, or 500 nucleotides in length.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid which has a non-naturally occurring sequence, or which has the sequence of part or all of a naturally occurring gene but is free of the genes that flank the naturally occurring gene of interest in the genome of the organism in which the gene of interest naturally occurs. The term therefore includes a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote. It also includes a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment. It also includes a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are mixtures of DNA molecules, vectors, or clones as they occur in a DNA library such as a cDNA or genomic DNA library. Also excluded are RNA molecules that consist of naturally occurring sequences (e.g., naturally occurring mRNA), except where the RNA is in a purified state such that it is at least 90% free of other naturally occurring RNA species. Thus, a naturally occurring mRNA in a whole mRNA preparation prepared from a cell would not be an xe2x80x9cisolated nucleic acid,xe2x80x9d but a single mRNA species purified to 90% homogeneity from that whole mRNA preparation would be.
As used herein, xe2x80x9cpercent identityxe2x80x9d of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410. 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic; acid molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nhn.nih.gov.
By xe2x80x9chybridizes under stringent conditionsxe2x80x9d is meant specific and non-covalent equilibrium binding by base-pairing to an immobilized reference nucleic acid in a hybridization solution containing 0.2xc3x97SSC (1.75 g/l NaCl, 0.88 g/l Na3citrate.2H2O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68xc2x0 C. Washings, if any are required to achieve equilibrium, are carried out with the hybridization solution.
A xe2x80x9cheterologous sequencexe2x80x9d is a nucleotide sequence that is not naturally operably linked to the OsGRP-A1 promoter in a naturally occurring organism.
A xe2x80x9cpromoterxe2x80x9d is a nucleotide sequence that is capable of directing transcription in at least one context, e.g., when it is operably linked to a heterologous sequence in a plasmid within a plant cell. In other words, a promoter can exist without downstream sequences to transcribe, so long as the promoter sequence can direct transcription when placed upstream of a heterologous sequence in a different context.
The sprout or seedling can be derived from monocots or dicots. As used herein, the term xe2x80x9csproutxe2x80x9d means a young shoot, including the cotyledon (as in dicots), scutellum (as in monocots), hypocotyl (the shoot below the cotyledon, as in bean), epicotyl (the shoot above the cotyledon, as in pea), coleoptile (as in cereal grains), and young root (radicle) grown from embryo of germinating seed. Germination begins with water uptake by the seed (imbibition) and ends with the start of elongation by the embryonic axis, usually the radicle. Therefore, germination does not include seedling growth, which commences when germination terminates. For germination to be completed, the radicle must expand and penetrate the surrounding structures. As used herein, the term xe2x80x9cseedlingxe2x80x9d means the juvenile plant grown from a sprout or a germinating seed, as defined in de Vogel, xe2x80x9cThe Seedling,xe2x80x9d In: Seedlings of Dicotyledons, Centre for Agricultural Publishing and Documentation, Wageningen, Netherlands, pp 9-25, 1983.
The promoter sequence of the invention can be introduced into a variety of plant expression vectors for expressing exogenous proteins in plant cells, transgenic plants, and seeds or embryos thereof. In addition, the isolated nucleic acids of the invention can be used as probes to isolate other promoters and/or genes that have the same expression pattern as e described herein (e.g., high level expression in seedling or embryo tissue). For example, nucleotides 1530 to 1630 (SEQ ID NO:6) or 1630 to 1730 (SEQ ID NO:7) of SEQ ID NO:1 can be used to screen genomic DNA libraries for genes that are regulated similarly to OsGRP-A1. Further, the methods of the invention can be used to produce transgenic plants or organs having specialized properties (e.g., longer shelf-life) as a consequence of expressing a heterologous RNA (e.g., a mRNA encoding an anti-ageing protein or an anti-sense RNA that inhibits expression of a senescence-associated gene) in a tissue or organ of a plant.
Further, nucleic acids containing SEQ ID NO:5 or fragments thereof can be used as a promoter (e.g., SEQ ID .NO:1) or as a probe for isolating gene promoters having the expression pattern for OsGRP-A1, as described herein.